Venoms of both sexes of Australian Northern (Missulena pruinosa) and Eastern (Missulena bradleyi) mouse spiders were studied in order to determine intersexual variations in venom yield, composition and bioactivity. Females of both species yielded more venom than males. High-performance liquid chromatography (HPLC) and mass spectrometry data further indicate a substantial degree of intersexual variation in the venom composition of both species. In a cricket (Acheta domestica) acute toxicity assay, only small intersexual differences were observed, but M. bradleyi venom was found to be considerably more potent than M. pruinosa venom. In the chick biventer cervicis nerve–muscle preparation, male but not female M. bradleyi venom induced large and sustained muscle contractions with fasciculation and decreased twitch height that could be reversed by CSL funnel-web spider antivenom. In contrast, venoms of both sexes of M. pruinosa did not induce significant effects in the chick biventer cervicis nerve–muscle preparation. We therefore conclude that female M. bradleyi venom and venoms from male and female M. pruinosa appear to contain few, if any, orthologs of δ-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These findings are consistent with clinical reports that mouse spiders, particularly species other than male M. bradleyi, do not appear to be a major medical problem in humans.